Project Summary The elicitation of broadly neutralizing antibodies (bNAbs) against HIV Env is one of the major challenges of HIV-1 vaccine development. As a vaccine component, HIV-1 Env however elicits limited neutralizing breadth and potency in most pre-clinical and clinical studies to date. The isolation of second generation of bNAbs from HIV-infected individuals, and characterization of their cognate epitopes on Env, offer a tremendous opportunity for understanding the mechanisms underlying the elicitation of bNAb responses. As one of the most functionally conserved and accessible elements of the HIV Env, the receptor binding site, namely CD4 binding site (CD4bs), plays a crucial role for virus engagement with receptor CD4 while serves as a vulnerable target for bNAb response in natural infections. VRC01-class bNAbs, a subset of CD4bs bNAbs isolated from donor 45 and numerous other HIV-infected individuals strongly supports the premise of utilizing VRC01-class bNAbs as vaccine template. However, current Env-based immunogens are not capable of eliciting potent VRC01-class bNAb response via vaccination. The inferred VRC01- class bNAb germline precursors (gVRC01) often display weak or no apparent affinity to prototypical Env immunogens. Thus, immunogen modifications including approaches for efficiently activating, selectively expanding, and precisely shepherding bNAb germline precursors (germline targeting) are needed for CD4bs bNAb elicitation, yet met with limited success. During the last funding period, we established a panel of Env-based designer immunogens with desirable antigenicity for eliciting CD4bs bNAb response. In this proposal, we aim to extend our effort by an innovative program consisting of the following three complementary specific aims: (1) to investigate the immunogenicity of novel immunogens/regimens in bNAb germline BCR knockin mice; (2) to investigate the immunogenicity of novel immunogens in guinea pigs and OmniMouse2 model that carries un- rearranged human antibody loci; and (3) to explore a novel neutralizing epitope in the C3/V4 region of the Env as potential bNAb response target. We believe that through this study we will (i) contribute to the thorough understanding of the basic aspects of the B cell response to the HIV-1 Env following vaccination and natural infection; and (ii) contribute new immunogens, new vaccine target, and optimized vaccination regimens leading to improved neutralizing responses targeting conserved Env elements.